Structural Bioinformatics Study of Five Transmembrane Rhomboid Proteases and Their AlphaFold 3-Predicted Water-Soluble QTY Analogues

Rhomboid proteases are integral membrane serine proteases involved in cellular signaling, membrane-protein quality control, mitochondrial homeostasis, and bacterial pathogenicity. However, their hydrophobic transmembrane domains make them difficult to solubilize and experimentally characterize without detergents, limiting structural and biochemical investigation. In this structural bioinformatics study, we applied the QTY code to five rhomboid proteases—Presenilin-associated rhomboid-like protein (PARL), Rhomboid-like protein 2 (RHBDL2), Rhomboid-like protein 4 (RHBDL4), Escherichia coli GlpG (ecGlpG), and Haemophilus influenzae GlpG (hiGlpG)—to design their water-soluble analogues. Within the transmembrane helices, leucine was replaced with glutamine, isoleucine and valine with threonine, and phenylalanine with tyrosine. We then used AlphaFold 3 to predict the structures of the native proteins and their corresponding QTY analogues. Despite transmembrane-sequence replacements ranging from 37.76% to 49.21%, structural superposition revealed remarkable structural agreement between all native–QTY pairs, with root mean square deviation values ranging from 0.251 to 0.450[Formula: see text]Å. The QTY analogues retained the characteristic [Formula: see text]-helical rhomboid architecture, while the calculated isoelectric points and molecular weights changed minimally. Molecular surface analysis further showed a substantial reduction in exposed hydrophobicity following QTY substitution while preserving the overall predicted molecular structures. These findings indicate that extensive hydrophobic-to-hydrophilic substitutions can be introduced into rhomboid transmembrane helices without substantially disrupting their predicted three-dimensional structures. The resulting QTY analogues provide candidate detergent-free proteins for future experimental characterization and may facilitate biochemical studies and therapeutic research involving medically important rhomboid proteases.